Site specific ligation of proteins to synthetic particles

ABSTRACT

The present invention relates to site-specific conjugation of synthetic particles to proteins.

[0001] This application is a continuation of U.S. application Ser. No.09/547,008, which claims priority to U.S. Provisional Patent ApplicationSerial No. 60/129,105, now abandoned.

FIELD OF INVENTION

[0002] The present invention relates to the field of site-specificconjugation of synthetic particles to proteins. Synthetic particlesinclude monodisperse synthetic particles, highly monodisperse nanoscaleparticles, and dendritic polymers (herein after dendrimers). Proteinsinclude four-helical bundle proteins and cytokines. The presentinvention also relates to both novel methods of conjugation of syntheticparticles to proteins and the resulting compositions.

BACKGROUND OF THE INVENTION

[0003] Biotechnology has made the production and engineering of proteinsa straight-forward commercially feasible technology. Recombinantproteins are linear amino acid polymers, synthesized from amino tocarboxy ends, that fold into three dimensional configurations during orafter synthesis. Biotechnology allows convenient production ofindividual protein molecules in bulk. Bioconjugate technology can add anadditional dimension to protein structures by allowing higher-orderorganization of the individual proteins using a synthetic framework.Advantages of higher order structures containing proteins include, undersome circumstances, therapeutically important properties including, butnot limited to, circulating half-life, tissue or intracellulartargeting, biodistribution, protein stability, ligand potency/activity,and protein immunogenicity. Regulating the relative positions andstoichiometries of individual proteins in higher order structures canalso give rise to wholly new activities and functions.

[0004] PEG (polyethylene glycol)is a synthetic material commonlyattached to proteins. A variety of means have been used to attach PEG toproteins. The most frequent method of attachment is through the aminogroups as found on the lysine residues or at the N-terminus (See PCTApplication WO 96/11953, incorporated by reference).

[0005] Kinstler et al., WO 96/11953 teaches that conjugation of PEG to aparticular protein, namely G-CSF, by conventional means has deficienciesrelated to the poor site-specificity of the PEG conjugation. Kinstleridentified that although the prior art provided multiple methods ofconjugating PEG to proteins, none of the methods known in the artallowed for selective attachment. As noted above, selective attachmentis desirable for many reasons including retention of proteinbioactivity. Kinstler identified a method of bioconjugation toselectively attach PEG to the N-terminus of G-CSF. The method disclosedin Kinstler is specific for the conjugation of G-CSF to PEG. It is notcontemplated by Kinstler, nor those skilled in the art, that the methodused to conjugate G-CSF to PEG can be used to conjugate other proteinsor to use different synthetic particles and materials while retainingparticular properties of the protein.

[0006] Thus, there is a need for a more universal method of conjugationand more particularly the use of better methods of conjugating proteinsto monodisperse, well-defined synthetic particles.

SUMMARY OF THE INVENTION

[0007] In its broadest sense, the present invention is directed toorthogonal chemistry for site specific conjugation/ligation of syntheticparticles to the N-terminus of proteins. Synthetic particles includemonodisperse synthetic particles, highly monodisperse nanoscaleparticles, and dentritic polymers (herein after dendrimers). Proteinsinclude four-helical bundle proteins and cytokines.

[0008] Dendrimers, a subset of highly monodisperse nanoscale particles(also known as dense star polymers), offer benefits that other carriersknown in the art lack. In particular, dendrimers exhibit moleculararchitecture characterized by regular dendritic branching with radialsymmetry. See U.S. Pat No. 5,527,524 incorporated by reference. Thisuniform architecture is desirable for homogenous ligated compositions.In addition, each dendrimer can be molecularly tailored to meetspecialized end uses by controlling the size, shape and properties ofthe dendrimer.

[0009] Cytokines are small proteins that engage cell-surface receptorsto elicit their biological activities. Synthetic particles are anyparticulate materials produced using synthetic chemical means.Monodisperse synthetic particles are any particulate materials producedusing synthetic chemical means that are limited to a single chemicalcomposition, size and architecture. Monodisperse nanoscale particles areany particulate materials produced using synthetic chemical means thatare limited to a single chemical composition, size and architecture andwhich measure between 1 and 999 nanometers in each dimension (ie.,length, width and depth).

[0010] One method of conjugation contemplated by the present inventionentails coupling of sulfhydryl-terminated synthetic particles with amaleimide spacer on the protein. Another contemplated method is based onusing a serine end-terminal (amine end) on the protein that can then beoxidized using periodate to form an aldehyde. The aldehydic-protein canthen be coupled to the synthetic functionalized with an aminooxyacetylgroup to form a stabilized oxime. A further method to achieveessentially the same result is to couple an amine-terminated syntheticparticle to the aldehydic protein. The Schiff's base that is formed canthen be stabilized using a mild reducing agent such as sodiumcyanoborohydride.

DETAILED DESCRIPTION OF THE INVENTION

[0011] One type of specialized synthetic particles are dendrimers.Dendrimers are polymers that are unimolecular assemblages possessing: 1)an initiator core; 2) interior layers (referred to as generations or G)made up of repeating-units, radially attached to the initiator core; and3) exterior surface of terminal functionality or terminal functionalgroups attached to the outermost generation. The size and shape of thedendrimer and the resulting functionality can be controlled by thechoice of the initiator core, the number of generations and the choiceof the repeating units employed at each generation. Since dendrimers canbe isolated at any particular generation, dendrimers can be obtainedhaving only the desired structural properties. (See U.S. Pat. No.5,527,524, incorporated by reference). Particular methods of producingdendrimers can be prepared according to methods described in U.S. Pat.No. 4,587,329, incorporated by reference.

[0012] PAMAM (polyamidoamine) dendrimers are microdomains which veryclosely mimic classical spherical micelles in shape, size, number ofsurface groups and area/surface groups. A significant difference betweenmicelles and PAMAM dendrimers is that PAMAM dendrimers are covalentlyfixed and robust compared to the dynamic equilibrating nature ofmicelles. This difference provides an advantage for the PAMAM dendrimersespecially when using them as encapsulation devices. This advantage ismost appreciated when the PAMAM dendrimers remain in the fifthgeneration or less. Generations more than five may cause congestion atthe surface.

[0013] Dendrimers suitable for this invention include those described inU.S. Pat Nos. 4,507,466, 4,558,120, 4,568,737, 4,587,329, and 5,527,524,incorporated by reference.

[0014] Although all synthetic particles are contemplated by thisinvention, monodisperse synthetic particles are more preferred. A morepreferred embodiment of the invention uses highly monodisperse nanoscaleparticles. An even more preferred embodiment uses PAMAM dendrimers.

[0015] Proteins for this invention are either joined directly to thesynthetic particle or in an alternative embodiment attached via alinker. In order to prepare the protein for the linker type applicationa GMBS-linker may be employed. A commercially obtained (Pierce,Rockford, Ill., USA) sulfo-GMBS (N-gamma-maleimidobutyryloxylsulfosuccinimide ester)linker sequence was appended to the N-terminalalanine of the protein at a high pH to achieve N-terminal sitespecificity of GMBS linker attachment. For the non-linker/directattachment method, proteins were engineered by standard biotechnologymethods to contain a serine at the N-terminus.

[0016] Although all proteins can potentially be utilized for thisinvention, a more preferred embodiment uses four-helical bundleproteins. An even more preferred embodiment uses cytokines. The proteinsused for this invention can be natural or recombinantly produced. A morepreferred embodiment uses recombinant proteins engineered for particularpurposes or bioactivity.

[0017] It is also contemplated that the synthetic particle or morespecifically a dendrimer can be engineered to be ligated to more thanone protein.

[0018] A preferred method for site specifically attaching a syntheticparticle to the N-terminus of a protein comprises the steps of:

[0019] 1) attaching a spacer on the N-terminus of a protein;

[0020] 2) forming a sulfhydryl on a synthetic particle at the amine; and

[0021] 3) combining said sulfhydrylized synthetic particle to saidspacer on the protein.

[0022] A more preferred method for site specifically attaching asynthetic particle to the N-terminus of a protein comprises the stepsof:

[0023] 1) converting a ser-terminated protein to an aldehyde;

[0024] 2) converting the amine of a synthetic particle to a oxiamine;and

[0025] 3)combining said aldehyde ser-terminated protein to saidsynthetic particle at said oxiamine.

[0026] The following three schemes describe processes of preparing novelconjugates. It would be obvious from the schemes to those skilled in theart that alternate reagents and modifications of the processes could beused depending on circumstances. These schemes are not intended to limitthe scope of the invention.

[0027] The following Examples are provided to illustrate the presentinvention and are not intended to limit the scope thereof. Those skilledin the art will readily understand that known variations of theconditions and processes of the following preparative procedures can beused to prepare these conjugates.

[0028] Without further elaboration, it is believed that one skilled inthe art can, using the, preceding descriptions, utilize the presentinvention to its fullest extent. Therefore, the following preferredspecific embodiments are to be construed as merely illustrative and notlimitative of the remainder of the disclosure in any way whatsoever.Compounds containing multiple variations of the structural modificationsillustrated in the preceding schemes or the following Examples are alsocontemplated.

[0029] The starting materials which are required for the above processesherein described are known in the literature or can be made by knownmethods from known starting materials.

EXAMPLE 1 EX. 1a

[0030] 0.1 mM of a cytokine (containing an N-terminal alanine residue)was reacted with sulfo-gmbs (γ-maleimido butyoloxy sulfosuccinimide)linker (0.2 mM) at pH 8.5 for 45 minutes, and then dialysed againstphosphate buffered saline (PBS, pH 7.2, Life Technologies, Inc,Gaithersberg, Md.).

EX. 1b

[0031] A 0.1 M solution of G-5 dendrimers were treated withnitrogen-purged 5 mM iminothiolane HC1, 1 MMEDTA in PBS to convert someof the dendrimer surface amines to sulfhydryls. After incubation for 45minutes with constant nitrogen purging, the reaction mixture wasdialysed against PBS.

EX. 1c

[0032] After dialysis, GMBS-linker cytokine and sulfhydryl G-5 dendrimerwere mixed (two parts GMBS-linker cytokine to one part G-5 dendrimersolution) and incubated for four hours at room temperature (rt).

EXAMPLE 2 EX. 2a

[0033] A cytokine containing an N-terminal serine was reacted with atwo-fold molar excess of sodium perodate for 15 minutes in 20 mMphosphate buffer (pH 7) to convert the epsilon amino group of theprotein to an aldehyde. The solution was then dialysed against 50 mMNaOAc (pH 4.5).

EX. 2b

[0034] Boc-aminooxyacetic acid was synthesized (Pochon et al., 1989) andmixed with G-5 dendrimer at 20-fold molar excess (relative to the numberof dendrimer surface amines in the presence of EDC (same molarconcentration as the Boc-aminooxyacetic acid) in PBS.

EX. 2c

[0035] After reacting overnight, the reaction mixture was dialysedagainst water. Dendrimer solution was concentrated u sing a Centricon-20(Amicon, Beverly, Mass.) and reacted with trifluoroacetic acid (TFA) for60 minutes at room temperature.

EX. 2d

[0036] Excess TFA was removed by exhaustive purging with nitrogen.Oxiamine dendrimers were resuspended and dialysed in water.

EX. 2e

[0037] The oxiamine dendrimers were mixed with the aldehyde Ser-cytokineat a molar ratio of 1:1 and incubated overnight at room temperature. Theconjugation reaction was then dialysed against 20 mM phosphate.

[0038] Structural Analysis

[0039] Trypsin Digestion of Cytokine Derivatives Modified for DendrimerConjugation

[0040] Dried sample was solubilized in 25 mM ammonium bicarbonate pH 7.8for a final concentration of 1.2 mg/ml. Trypsin (Promega, Madison, Wis.,USA) was added at a 1:60 (w/w) enzyme to substrate ratio and incubatedfor 6 hours at ambient temperature. Sample was mixed with matrix (33 mMalpha-cyano-4 hydroxycinnamic acid—Hewlett Packard, Palo Alto, Calif.,USA) at a 1:8 (v/v) ratio. Approximately 8 pmole of digest was analysedby MALDI-TOF (Matrix assisted laser desorption ionization-time offlight).

[0041] Matrix Assisted Laser Desorption Ionization-Time of FlightAnalysis

[0042] MALDI analysis was performed on untreated cytokine of Ex. 1a, theproduct of Ex. 1a and the product of Ex. 2a using a PerSeptive BiosystemVoyager—DERP Benchtop MALDI-TOF Mass Spectrometer (Framingham, Mass.,USA) with a 1.3 meter flight tube. Samples were run in the linear modewith delayed extraction using alpha-cyano-4 hydroxycinnamic acid matrix.Both trypsin digested (see above) and undigested samples were analyzed.

[0043] Untreated Cytokine of Ex. 1a and Untreated Cytokine of Ex. 2awere Conjugated to Dendrimers Site-Specifically:

[0044] Attempts to identify a matrix suitable to volatilizedendrimer-untreated cytokine conjugates for MALDI TOF analysis were notsuccessful (Data not shown). Therefore, MALDI-TOF analysis of theproduct of Ex. 1a and the product of Ex. 2a were used to demonstrate thesite-specificity of protein modification (N-terminal GMBS linkeraddition or N-terminal oxidation to aldehyde group) for coupling, andtherefore of dendrimer-protein conjugation.

[0045] Analysis of untreated cytokine of Ex. 1a (FIG. I) reveals asingle predominant peak at the predicted average mass, with minor peakoccurring at about at 20 AMU higher molecular weight. Addition of theproduct of Ex. 1a to untreated cytokine of Ex. 1a increases its mass by169 AMU (FIG. II): this species is detected in the chromatograph (FIG.II), though substantial amounts of the starting materials remain. In thecase of untreated cytokine of Ex. 2a oxidized to have an N-terminalaldehyde, cytokine molecular weight is reduced by about 31 AMU (FIG.III), as was predicted. A minor band of unoxidized cytokine of Ex. 2a isalso detected. These data indicate that significant fractions of theparent proteins were converted to conjugation-ready form and that theconjugation-ready forms of the proteins contain primarily a singlemodification per protein molecule.

[0046] FIGS. I, II and III. MALDI analysis of untreated cytokine of Ex.1a species activated for dendrimer conjugation.

[0047] FIG. I: Unactivated untreated cytokine of Ex. 1a.

[0048] FIG. II: Product of Ex 1a. (Activated for maleimide/sulfhydrylconjugation); and

[0049] FIG. III: Product of 2a. (activated for oxiamine conjugation)

[0050] Analysis of tryptic digest fragments of untreated cytokine of Ex.1a modified for conjugation was used to localize the position ofmodification for conjugation. Sites of trypsin cleavage are shown inFIG. IV and predicted masses of digest fragments are presented in FIG.V. MALDI-TOF chromatographs of tryptic digests of the product of Ex 1aand the product of Ex. 2a are shown in FIG. IV and FIG. V respectively.With the exception of low molecular weight fragments (T-3, T-5 andT-10), predicted tryptic digestion fragments can be accounted for bothchromatographs. Note that because the experiment was not performed underreducing conditions, fragments T-1 and T-7 appear together asdisulfide-linked peptides.

[0051] In the case of the product of Ex 1a, addition of the linker isexpected to increase the mass of the T-1-T-7 linked peptides by 169:this fragment is not detected in the chromatograph, though a fragmentcorresponding to the T-1-1-7 peptide plus 184-185 AMU is (FIG. IV).Presumably, this is due to an adduct that was formed during the trypticdigest. Nonetheless, sufficient additional mass to account for the GMBSlinker appended to untreated cytokine of Ex. 1a (FIG. IV) is notassociated with other tryptic fragments, so the results are consistentwith the interpretation that a single GMBS linker is linked to the N endof untreated cytokine of Ex. 1a.

[0052] The mass of T-1-T-7 fragment of product of Ex. 2a should be3,246.8 (FIG. V), and such a fragment is present in the chromatograph.Unoxidized T-1-T-7 can also be detected.

[0053] Thus, oxidation of untreated cytokine of Ex. 2a and GMBS linkeraddition to untreated cytokine of Ex. 1a can be detected by MALDI-TOF.In both cases, the cytokines are predominantly modified once permolecule, and the modifications are localized to the T-1-T-7 trypsindigestion fragments. These data support the notion that conjugation todendrimers occured at the N-terminus of the cytokine molecules, asdesired.

[0054] FIG. IV and V. Maldi-TOF analysis of trypsin digests of cytokineactivated for coupling to dendrimers.

[0055] FIG. IV: The product of Ex. 1a (For sulfhydryl/maleimideconjugation) and

[0056] FIG. V: The product of Ex. 2a (For oxiamine conjugation. FragmentMW (daltons) T-1 (untreated cytokine of Ex. 1a) 1738.09 T-1 (product ofEx. 2a) 1723.09 T-1 (untreated cytokine of Ex. 2a) 1754.09 T-2 2605.94T-3 402.47 T-4 1075.26 T-5 317.41 T-6 1400.58 T-7 1526.76 T-8 720.94 T-91009.07 T-10 276.31 T-11 1812.03

[0057] Predicted masses of tryptic fragments of untreated cytokine ofEx. 1a, untreated cytokine of Ex. 2a and their derivatives used indendrimer conjugation. Note that the T-1 and T-7 fragments are disulfidelinked. In the end product of Ex 2a the predicted combined mass of T-1and T-7 is 3,246.8.

[0058] Functional Analaysis

[0059] Quantitative Amino Acid Analysis of Proteins and Conjugates:

[0060] Quantitative amino acid analysis (Moore and Stein,1963) was usedto quantify the protein concentration of untreated cytokine-dendrimerconjugate solutions. Protein-dendrimer conjugates were subjected tovapor phase acid hydrolysis using 6N HCl (with 1% phenol added) for 1.5hours at 150° C. (Bidlingmeyer et al. 1984). A Water's Pico-Tagworkstation (Waters, Midford, Mass., USA) was used for the hydrolysis.The resulting hydrolysate was analyzed by post-column ninhydrin aminoacid analysis on a Beckman model 6300 amino acid analyzer (Beckman, Inc,Palo Alto, Calif., USA).

[0061] hIL-3 Receptor Binding Assay

[0062] Baby hamster kidney (BHK) cells which had been stably transfectedwith the gene for the IL-3 receptor subunit (BHK″) isolated from AML193.1.3 cells were used in receptor binding studies (Thomas et al.,1995). The transfected BHK″ cells were grown in Dulbecco's ModifiedEagle Media, 5% FBS, 2 mM glutamine and harvested using CellDissociation Media (Sigma Chemical Company; St. Louis, Mo). The BHKUcells were washed and then resuspended in assay media (Iscove's ModifiedDulbecco's Media, 5% FBS, 0.2% sodium azide) and kept frozen at −20° C.until their use in the binding assays. Assay incubations (0.1 ml),containing the radioligand (radio labeled hIL-3 receptor agonist;300-500 pM) and competing compound were performed at 0-4° C. andinitiated by the addition of ice cold cells. Incubations were halted byseparation of bound and free radiolabel by rapid centrifugation (12000×gfor one min) in an Eppendorf 5415C centrifuge. The supernatant wasaspirated and the tube tip containing the cell pellet with theassociated radioactivity cut off and counted in an ICN MicromedicsSystem automatic gamma counter. Nonspecific binding was defined as theresidual binding occurring in the presence of excess standard hIL-3receptor agonist (1000 nM). Radio labeled hIL-3 receptor agonistcompetition binding experiments were analyzed using Scatchard and Hilltransformations and the IC₅₀ values for compounds determined usinglogit-log analysis. The radio labeled hIL-3 receptor agonist(¹²⁵I], afully bioactive hIL-3 receptor agonist with a 14 amino acid N-terminalextension to facilitate radioiodination (Thomas et al. 1995), was usedin these experiments was iodinated by the lactoperoxidase method and hadspecific activities of 500-2000 Ci/mmol as measured by ELISA or selfdisplacement analysis.

[0063] Untreated Cytokine of Ex. 1a Conjugated to Dendrimers Recognizethe hIL-3 Receptor:

[0064] Receptor binding experiments revealed that bothdendrimer-cytokine conjugates retained the ability to recognize thehIl-3 receptor alpha subunit. The affinity of untreated cytokine of Ex.2a and the product of Ex. 1a were comparable, and their receptor bindingwas two to fourfold less avid than unmodified untreated cytokine of Ex.1a (W. Hood, unpublished, B. Klein et al, manuscript in preparation).Both conjugates exhibited modest decreases in receptor affinity relativeto the corresponding precursor molecule, with conjugate binding aboutfourfold less avid than free protein. Sample IC₅₀ (nM) Product of Ex. 1a5.4 ± 1.2 Sulfhydryl/maleimide 24.0 ± 1.6  conjugate Untreated cytokineof Ex. 2a 4.45 ± 0.8  Oxime conjugate 17.0 ± 1.5 

[0065] Affinities of cytokine species and their dendrimer conjugates forthe hIL alpha receptor subunit. IC₅₀ values were calculated in nMuntreated cytokine of Ex. 1a of the conjugate as determined by aminoacid composition analysis. The presented affinities are the mean ofthree independent determinations, and standard error values (SE) areshown.

We claim:
 1. A substantially homogenous conjugate composed of asynthetic particle attached to the N-terminous of a protein.
 2. Theconjugate of claim 1 wherein the synthetic particle is a monodispersesynthetic particle.
 3. The conjugate of claim 1 wherein the syntheticparticle is a monodisperse nanoscale particle.
 4. The conjugate of claim1 wherein the synthetic particle is a dendrimer.
 5. The conjugate ofclaim 1 wherein the synthetic particle is a PAMAM dendrimer.
 6. Theconjugate of claim 1, 2, 3 or 4 wherein the protein is a four-helicalbundle protein.
 7. The conjugate of claim 1, 2, 3 or 4 wherein theprotein is a cytokine.
 8. A pharmaceutical composition of the conjugateof claim 1, 2, 3, 4, 5, 6 or
 7. 9. A method for site specificallyattaching a synthetic particle to the N-terminus of a protein, saidmethod comprising the steps of: a) attaching a spacer on the N-terminusof a protein; b) forming a sulfhydryl on a synthetic particle at theamine; and c) combining said sulfhydrylized synthetic particle to saidspacer on the protein.
 10. A method for site specifically attaching asynthetic particle to the N-terminus of a protein, said methodcomprising the steps of: a) converting a ser-terminated protein to analdehyde; b) converting the amine of a synthetic particle to a oxiamine;and c) combining said aldehyde ser-terminated protein to said syntheticparticle at said oxiamine.
 11. The method of claim 9 or 10 wherein thesynthetic particle is a monodisperse synthetic particle.
 12. The methodof claim 9 or 10 wherein the synthetic particle is a PAMAM dendrimer.13. The method of claim 9 or 10 wherein the synthetic particle is ahighly monodisperse nanoscale particle.
 14. The method of claim 9 or 10wherein the synthetic particle is a dendrimer.
 15. The method of claim9, 10, 11, 12, 13 or 14 wherein the protein is a four-helical bundleprotein.
 16. The method of claim 9, 10, 11, 12, 13 or 14 wherein theprotein is a cytokine.